A major chromosomal abnormality is detected in approximately 1 of 140 live births1 and in a much higher fraction of fetuses that do not reach term or are still-born2. The most common aneuploidy is trisomy 21, which currently occurs in 1 of 730 births1. Though less common than trisomy 21, trisomy 18 (Edwards Syndrome) and trisomy 13 (Patau syndrome) occur in 1 in 5,500 and 1 in 17,200 live births, respectively1. A large variety of congenital defects, growth deficiencies, and intellectual disabilities are found in children with chromosomal aneuploidies, and these present life-long challenges to families and societies3. For these reasons, much effort has been devoted to detecting chromosome abnormalities during early fetal life, at a time when therapeutic abortions can be offered as an option to prospective parents.
There are a variety of prenatal diagnostic tests that can indicate increased risk for fetal aneuploidy, although invasive tests such as amniocentesis or chorionic villus sampling are the current gold standard4 and are associated with a non-negligible risk of fetal loss. More reliable, non-invasive tests for fetal aneuploidy have therefore long been sought. The most promising of these are based on the detection of fetal DNA in maternal plasma, as pioneered by Lo's group5. It has been demonstrated that massively parallel sequencing of libraries generated from maternal plasma can reliably detect chromosome 21 abnormalities6,7. In the most comprehensive study to date8, 98.6% of fetuses with trisomy 21 were detected in maternal plasma, with a false positive rate of 0.2 percent. In an additional 0.8 percent of samples, the test failed to give a result. These exciting studies promise a new era of non-invasive prenatal testing.
Currently, almost half of trisomy 21 babies are born to mothers less than 35 years of age, as more than 80% of pregnant women are under 359,10. Though the risk of invasive procedures is thought to outweigh the benefit of invasive testing for eligible young mothers, it is clear that the vast majority of births associated with chromosomal aneuploidies could be safely prevented with reliable non-invasive tests that could be safely administered to all pregnant women. Prenatal testing is an extraordinarily stressful exercise for pregnant mothers and their families, and the more rapid the process, the better.
To achieve this goal with circulating fetal DNA testing, decreases in cost and increases in throughput will be necessary. There are three major components of plasma DNA testing: preparation of DNA libraries for loading on the sequencing instrument, the sequencing of these libraries, and their analysis. The second component is being addressed by instrument manufacturers, who have made remarkable progress over the last few years. Potential improvements in the first and third components are the subject of the current study.
The only commercially available test for circulating fetal DNA aneuploidy involves the preparation of whole genome libraries and the analysis of a sufficient number of sequences on the relevant chromosomes to reliably detect small differences in copy number. The preparation of whole genome libraries involves several sequential steps, including end-repair, 5′-phosphorlyation, addition of a terminal dA nucleotide to the 3′ ends of the fragments, ligation of the fragments to adapters, and PCR amplification of the ligated products, many of which require intervening purifications. The PCR products are then quantified and loaded on the sequencing instrument. Following the sequencing run, the tags are aligned to the human genome and assessed with Digital Karyotyping11, i.e., the number of tags per genomic locus is used to construct a virtual karyotype. Another recently described test involves fewer, but still a large number of, steps to prepare libraries for sequencing12.
There is a continuing need in the art to rapidly and non-invasively detect genetic abnormalities.